title: Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.
abstract: Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.
authors.affiliation: Novartis Vaccines and Diagnostics Research Center, Via Fiorentina 1, Siena 53100, Italy.
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Gentile
authors.forename: Sara
authors.initials: S
authors.lastname: Melchiorre
authors.forename: Carla
authors.initials: C
authors.lastname: Emolo
authors.forename: Monica
authors.initials: M
authors.lastname: Moschioni
authors.forename: Claudia
authors.initials: C
authors.lastname: Gianfaldoni
authors.forename: Laura
authors.initials: L
authors.lastname: Pancotto
authors.forename: Ilaria
authors.initials: I
authors.lastname: Ferlenghi
authors.forename: Maria
authors.initials: M
authors.lastname: Scarselli
authors.forename: Werner
authors.initials: W
authors.lastname: Pansegrau
authors.forename: Daniele
authors.initials: D
authors.lastname: Veggi
authors.forename: Marcello
authors.initials: M
authors.lastname: Merola
authors.forename: Francesca
authors.initials: F
authors.lastname: Cantini
authors.forename: Paolo
authors.initials: P
authors.lastname: Ruggiero
authors.forename: Lucia
authors.initials: L
authors.lastname: Banci
authors.forename: Vega
authors.initials: V
authors.lastname: Masignani
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002448:Cell Adhesion; D004195:Disease Models, Animal; D000939:Epitopes / Q000737:chemistry; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018805:Sepsis / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D010455:Peptides; D036781:Fimbriae Proteins